Previous work indicated the value of combining a targeting element having a high affinity for a cell surface receptor and an activity element having a lower affinity for a second cell surface receptor through which signaling occurs (for example by fusion of protein domains via a linker). A mutation may be introduced into the activity element to reduce its receptor binding so that its binding affinity is below that of the targeting element for its receptor. This approach has been shown to enhance the specificity of an activity element for target cells relative to side effect cells by as much as 20-fold, with some specificity enhancement attributable to the attached targeting element and the activity-reducing mutation.
However, in some therapeutic settings, the desired target cell is much less accessible than cells through which adverse side effects are mediated. For example, solid tumors often lack lymph node drainage, therefore therapeutic proteins that enter from the circulation only perfuse the tumor by diffusion. This results in the concentration of the therapeutic fusion protein being many times greater in the vicinity of side-effect cells, such as normal tissue, compared to target cells, such as tumor cells. In addition to tumors, targeting of proteins to the brain also results in limited access by therapeutic proteins due to the blood-brain barrier. Therefore, there is a need in the art for improved approaches to targeting of therapeutic protein activities, as well as for therapeutic proteins with enhanced specificity for their target cells.